Journal of Food Engineering 116 (2013) 588–597

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Journal of Food Engineering

journal homepage: www.elsevier.com/locate/jfoodeng

Preparation and characterization of glycerol plasticized (high-amylose)

starch–chitosan films
Huihua Liu a,⇑, Raju Adhikari b, Qipeng Guo c, Benu Adhikari a
a
School of Health Sciences, University of Ballarat, Ballarat, Victoria, Australia
b
CSIRO Materials Science and Engineering, Clayton South, Victoria 3169, Australia
c
Polymers Research Group, Institute for Frontier Materials, Deakin University, Locked Bag 2000, Geelong, Victoria 3220, Australia

a r t i c l e i n f o a b s t r a c t

Article history: The chitosan–starch (high amylose) blend film (1;1), via microfluidization, was prepared by casting with
Received 19 September 2012 different glycerol concentration (0%, 2.5%, 5% and 10%). The films were characterized for their mechanical,
Received in revised form 4 December 2012 thermal and morphological properties. The addition of glycerol at 5% (w/w) and higher concentrations
Accepted 24 December 2012
resulted in decrease in tensile strength, increase in elongation at break due to plasticization. The well-
Available online 31 December 2012
known antiplasticization was observed in the polymer films with 2.5% of glycerol. The addition of glycerol
promoted the interactions among chitosan, starch and glycerol through hydrogen bonding as reflected on
Keywords:
the shifting of main peaks of the glycerol-free film to higher wavenumbers as shown by FTIR spectra. The
Chitosan
Starch
decrease in intensity of glycerol-related peaks in starch–chitosan–glycerol films in both 1H NMR and 13C
FTIR NMR spectra proved the strong interactions (decrease in glycerol mobility) occurring among starch,
NMR chitosan and glycerol in glycerol-plasticized films.
Microfluidization Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction bility, biodegradable nature and anti-bacterial characteristics.

There have been sustained research interests in developing
starch-based biocomposite films in response to the increasing de-
mands on environmental-friendly packaging material. Starch is
one of the most preferred green packaging materials due to its ra-
pid biodegradable nature, having renewable source and availability
in relatively low cost (Liu et al., 2011a,b; Majdzadeh-Ardakani
et al., 2010; Nejad et al., 2011; Qiao et al., 2010). However, pure-
starch has a relatively high glass transition temperature (Tg) value
within the moisture content of interest (15%, w/w) and is sensi-
tive to humidity (Myllärinen et al., 2002) both of which greatly
limit the utilization of starch-based biodegradable materials. The
addition of plasticizers can improve the flexibility of the pure
starch films and glycerol is commonly recognized as one of the
most suitable plasticizers (Cervera et al., 2004). Chitosan is also
attracting strong research interest as it is nontoxic, biodegradable
and biocompatible cationic polysaccharide (Mathew et al., 2006).
This biopolymer has been quite extensively used in tissue engi-
neering (Di Martino et al., 2005; Mi et al., 2001), scaffolding
(Mao et al., 2003), and as biofunctional materials (García et al.,
1998). The common application of chitosan is attributed to its pre-
mium film-forming ability, strong mechanical strength and flexi-
⇑ Corresponding author. Tel.: +61 3 5327 9942; fax: +61 5327 9240.
E-mail addresses: h.liu@ballarat.edu.au (H. Liu), raju.Adhikari@csiro.au
(R. Adhikari), qguo@deakin.edu.au (Q. Guo), b.adhikari@ballarat.edu.au (B. Adhikari).
Considering the advantages and unique characteristics of starch
and chitosan, it is expected that a blend of chitosan/starch would
be able to form a biodegradable film that would shows improved
mechanical properties (better strength and flexibility), lower water
permeability and antibacterial properties (Bangyekan et al., 2006;
Mathew and Abraham, 2008) compared to starch-based films
without chitosan. Moreover, microfluidization is a well-docu-
mented promising high energy homogenization method that had
been widely applied in the emulsion preparing process in various
area including personal care, cosmetics, health care, pharmaceuti-
cals, and agrochemicals, to improve stability of the emulsion attri-
bute to its ability to produce exceptionally fine and stable emulsion
(Jafari et al., 2007). During microfluidiziation, two steam of flow
collide with each other at a pressure up to 150 MPa and force them
through microchannels toward an impingement area creates a tre-
mendous shearing action (Salvia-Trujillo et al., 2012). Therefore,
microfluidization had been applied in the current study with the
aim to further improve the miscibility of chitosan and starch.
There are considerable numbers of studies which have investi-
gated different aspects of low amylose (LA) starch–chitosan films
(Bourtoom and Chinnan, 2008; Chillo et al., 2008; Dutta et al.,
2009; Rojas-Graü et al., 2006; Vicentini et al., 2005). Antibacterial
membranes were prepared from a mixture of hydrolyzed starch.
The effect of the ratio of chitosan and starch on the properties
(water vapor permeability, mechanical properties and miscibility
of biodegradable blend etc.) of the starch/chitosan films had been

0260-8774/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jfoodeng.2012.12.037
 H. Liu et al. / Journal of Food Engineering 116 (2013) 588–597
investigated in Bourtoom and Chinnan (2008)’s work. It was found
that the increase in the plasticizer concentration resulted in a de-
crease in tensile strength while the elongation at break, water va-
por permeability and film solubility were increased (Bourtoom and
Chinnan, 2008).
Actually, in literature, it had been well documented that the re-
crystallization and retrogradation process is closely related to the
amount of amylose and amylopectin presented. In the high-amy-
lose starch, both the re-crystallization and retrogradation are
greatly suppressed due to the lack of the branch structure amylo-
pectin; therefore, the film-formation ability and the stability of the
film are enhanced in high-amylose starch films (Fredriksson et al.,
1998; Rindlav-Westling et al., 2002). Recently, the film forming
behavior of low and high amylose had been studied by one of
the author (Muscat et al., 2011) and it had been shown that HA
films exhibited higher glass transition temperature, high tensile
strength, higher modulus of elasticity and lower elongation at
break than those obtained from LA starch. Therefore, we believe
that in such context, biodegradable film with excellent properties
could be produced using the HA starch. For instance, Pelissari
et al. reported the combined effects of the chitosan and low amy-
lose starch polysaccharides on the film physical properties such
as mechanical properties and water vapor permeability. The
authors also reported the application of constrained mixture de-
sign method on understanding the interplay effect of starch, chito-
san and glycerol. It is found the addition of relative small amount
of chitosan increase the mechanical resistance and decreased the
water vapor permeability (WVP) of the film (Pelissari et al., 2012).
However, the effect of plasticizer concentration was not taken
into consideration in the mentioned study. In addition, the crystal-
lization properties of the high-amylose starch–chitosan films at
different plasticizer concentration are not well documented in lit-
eratures. In this context, the objectives of this work were to inves-
tigate the effect of glycerol concentration on the interaction of
amylose and chitosan in terms of the mechanical properties, sur-
face morphology, thermal properties and molecular mobility char-
acteristics in the starch–chitosan films.
2. Materials and methods
2.1. Materials
High amylose (HA) cornstarch with amylose: amylopectin ratio
of (80:20) and moisture content of 12.60% (w/w) was purchased
through Tim Stock, Ballarat. Glycerol was used as plasticizer and
purchased from Consolidated Chemical Company, Melbourne, Vic-
toria. All the materials were used as received and the moisture con-
tent of the raw materials was compensated for while preparing the
solution for starch gelatinization. Chitosan (448877-Medium
molecular weight) was purchased from Sigma Aldrich (NSW,
Australia).
2.2. Methods
2.2.1. Gelatinization of starch–water slurry
The starch–water slurry was prepared by suspending 5% (w/w)
starch in deionized water. The gelatinization of this slurry was car-
ried out using a high temperature–high pressure laboratory reactor
(Amar Equipment Company, Mumbai, India). The slurry was
heated to 140 °C using 600 rpm agitator speed. The solution was
held for 30 min at 140 °C before cooling down to 90 °C maintaining
the same rotational speed. The gelatinization time (30 min) was
chosen to allow complete gelatinization of the starch. The com-
plete gelatinization was ascertained by using a polarized light
microscope (Ernest Leite Wetzlar, Germany, fitted with Q-Imaging
camera).
589
2.2.2. Preparation of chitosan solution
An aqueous solution (2%, w/w) of chitosan was prepared by dis-
solving chitosan powder into distilled water containing 0.5% (w/w)
acetic acid. Firstly, 0.5% acetic acid-water solution was prepared.
Subsequently, under agitation, 2 g chitosan was slowly added into
98 ml water-acetic acid solution. The mixture (chitosan/acetic
acid/water) was continuously stirred overnight to obtain fine and
clear chitosan solution.
2.2.3. Film casting and conditioning
The gelatinized high-amylose starch and chitosan solutions
were mixed in a way that the 1:1 (w/w) starch:chitosan solution
ratio was maintained. This mixture was further mixed with various
concentration of glycerol (2.5%, 5% and 10%, w/w) with constant
agitation. The starch–chitosan–glycerol mixture was subjected to
homogenization with a microfluidizing Processors (M-110L Pneu-
matic, Newton, USA) at an operating pressure of 80 MPa. Five re-
peated passes were conducted in order to obtain a fine solution.
Films were prepared by syringing 15 mL fully homogenized solu-
tion into 85 mm plastic polystyrene dishes. The cast solutions were
dried overnight at 65 ± 1 °C in an oven to obtain the films. Finally,
all the films were conditioned in a desiccator containing lithium
chloride (11.3% RH at 25 ± 1 °C) for at least 48 h before analysis.
2.2.4. Moisture content and particle size profile
The moisture content of the prepared films was determined by
a gravimetric method, whereby samples were dried at 105 ± 0.5 °C
in a laboratory oven (UNE PA, Memmert, Germany) until constant
weight was achieved (Jiang et al., 2010). Approximately 1.0 g film
samples were placed in previously dried and cooled glass petri dish
and kept in the oven for 8 h. Weights of the samples were taken be-
fore and after drying using a digital balance (±0.1 mg), (AE200S,
Mettler, Switzerland). Tests were conducted in triplicates and the
average values are reported.
2.2.5. The surface morphology
Surface morphology of prepared film samples was studied by
using a scanning electron microscope (JSM 6300 SEM, JEOL, Tokyo,
Japan). Prepared film samples were directly deposited on alumi-
num stubs using double-sided adhesive carbon conductive tape
and were coated with a thin gold layer with the help of gold sput-
ter. An accelerating potential of 12 kV was used during
experiments.
2.2.6. Measurement of crystallinity
X-ray diffraction (XRD) pattern of the prepared film samples
was measured using a Siemens (D501, Siemens, Karlsruhe, Ger-
many) diffractometer operating at 40 kV and 40 Ma with Cu Ka
radiation (a = 1.54 Å). Diffractograms were taken between 5° and
35° (2h) at a rate of 1.20°/min (2h) and with a step size of 0.05°
(2h). The % crystallinity was measured by using the Trace 6.0 soft-
ware package associated with the hardware following the method
proposed by Sottys et al. (1984).
2.2.7. Attenuated total reflectance–Fourier transform infrared analysis
(ATR–FTIR)
The IR spectra of the samples were measured using a Fourier
transform infrared (FTIR) spectrometer (Model 400, Perkin Elmer,
Australia) in the wavenumber range of 4000–600 cm1 with the
average of 64 scans. The resolution and signal to noise ratio (SN)
are 0.4 cm1 and 15,000:1, respectively, as provided by the sup-
pler. 0.1 g of sample was used for each test and each sample was
vacuum sealed in a water-resistant polyethylene pack and stored
in a container containing silica gel desiccant before the IR tests.
590 H. Liu et al. / Journal of Food Engineering 116 (2013) 588–597
2.2.8. Thermal properties
The thermal property of the prepared film samples was deter-
mined by DSC thermograms. The samples were analyzed using
Mettler DSC-028 (Mettler Toledo, USA). The system was calibrated
for temperature and heat flux with indium (melting point 156.6 °C,
heat of fusion 3.28 kJ mol1). The samples (6–15 mg) were
weighed in aluminum pans and hermetically sealed and an empty
pan was used as the reference. Scans were carried out over the
temperature range of 0–160 °C at a heating rate of 10 °C min1 un-
der nitrogen. The Tg was determined at onset of the endothermic
shift in the heat flow curve.
2.2.9. Nuclear magnetic resonance (NMR)
Film sample was dissolved in DMSO-d6, which is accepted to be
one of the most popular solvents in solution NMR measurements.
Before proceeding with the NMR measurement, 10 mg of film sam-
ple was dissolved in 1 ml DMSO-d6 for 10 min. The 1H was ob-
tained from Bruker 500. The films without glycerol were found to
be harder to dissolve compared to glycerol-containing films. In this
context, the films having glycerol were kept in oven (60 °C) for 1 h
to completely dissolve the sample before NMR test.
2.2.10. Statistical analysis
Triplicate runs were carried out for each experiment and the
data were subjected to statistical analysis. One-way analysis of
variance (ANOVA) was carried out to evaluate the significance in
differences (p < 0.05). SPSS Version 10.0 software (SPSS Inc., Chi-
cago, IL) was used for this purpose. Data are presented as mean
and standard deviation of triplicate analyses.
3. Results and discussion
3.1. The film appearance
The starch–chitosan films showed much lower extent of crack-
ing and shrinkage compared to starch-only films. The starch-only
films were brittle and showed higher degree of cracking and
shrinkage. This observation is consistent with the observations
made earlier by Muscat et al. that the plain starch film showed
brittleness and had a tendency of cracking even when the films
were dried at ambient temperature (Muscat et al., 2011). On the
other hand, the addition of chitosan resulted into intact, smooth
films which could be easily peeled off from the drying plastic
dishes. This clearly indicated the improved compatibility of chito-
san and starch under the prevailing drying condition. The presence
of chitosan suppressed the cracking and shrinkage which occurred
in starch-only films.
3.2. Mechanical properties
The mechanical properties (tensile strength, modulus of elastic-
ity and elongation at break) and thickness of films containing glyc-
erol are summarized in Table 1. The starch-only films were rigid
and brittle as mentioned; also the blend of starch and chitosan
films in the absence of glycerol showed brittleness. On the other
Table 1
Mechanical properties (tensile strength (TS), modulus of elasticity (EM), elongation at break (E)), thickness, Tg and melting point (TM) of the starch–chitosan films.
Sample ID TS (MPa) EM (MPa)
No glycerol – –
2.5% Glycerol 19.007 ± 4.379 9.074 ± 1.477
5.0% Glycerol 3.443 ± 0.620 2.868 ± 0.627
10.0% Glycerol 1.332 ± 0.049 1.824 ± 0.195
Plain starch – –
1% Chitosan – –
hand, the typical effects of addition of plasticizers to polysaccha-
ride system on mechanical properties, such as an increase in elon-
gation in break and decrease in tensile strength (Garcia et al.,
2006), are observed in the glycerol-containing samples (Table 1).
The elongation at break values for glycerol plasticized samples
are 13.5%, 14.0%, and 18.0% at 2.5%, 5.0% and 10.0% (w/w) glycerol
concentrations, respectively. The increased flexibility of the films
at higher plasticizer concentration can be attributed to interaction
of plasticizer–polymer chains which facilitated the sliding of chain
and thus help to improve the overall flexibility and the chain
mobility. This postulation is further supported by the NMR results
presented in Section 3.7.
It is worth mentioning here that the film containing 2.5% glyc-
erol exhibited the highest tensile strength and the lowest elonga-
tion at break values. This observation suggested that the
commonly observed plasticization effect was not seen in this sam-
ple. This behavior might be due to ‘antiplasticization’ effect of glyc-
erol when its concentration is lower than 12% (w/w) (Lourdin
et al., 1997a,b). The antiplasticization effect of glycerol had been
well discussed in Lourdin et al.’s work, where they concluded this
behavior to be a ‘cross-linker’ effect which led to increased crystal-
linity and greater rigidity in the starch–glycerol films. Specifically,
the strong interactions between glycerol and starch biopolymer led
to the so-called ‘cross-linker’ effect which resulted in the decrease
in the free volume of the system and reduced molecular mobility of
the polymer chains. Furthermore, the reorganization of the poly-
meric network due to strong polymer-plasticizer interactions fa-
vored the crystallization process, ultimately leading to the
increased chain stiffness and decreased flexibility of the material
(Lourdin et al., 1997a,b). This observation is supported by the
XRD results presented in the ensuing section.
3.3. The rubbery and crystalline nature of the films
The XRD patterns of starch-only, pure chitosan and starch–
chitosan blend films at different glycerol concentrations are pre-
sented in Fig. 1, where the diffraction patterns are offset to avoid
the overlapping of the patterns. The main peak positions of each
sample are summarized in Table 2.
As shown in Fig. 1, all the samples showed different extent of
crystallinity. The Chitosan-only film matrix was mostly amorphous
in nature with very small crystalline fraction imbedded in the
amorphous matrix as indicated by small hump at 2h = 9.4°. This
could be due to the intra-molecular interactions between NHþ 3
and hydroxyl groups that led to the formation of intramolecular
hydrogen bonding which limited the movement of the chitosan
chain (Mathew et al., 2006). The diffraction pattern of the plain
starch showed peaks around 5.9°, 14.95°, 17.6°, 19.65° and 22.2°
which correspond to the typical B-type starch diffraction pattern
(BeMiller and Whistler, 2009). The addition of chitosan to plain
starch decreased the crystallinity of the film as reflected in the
lower diffraction peaks at (5.9°, 17.6° and 19.65°). Furthermore,
the characteristic XRD peak of chitosan (9.4°) did not appear in
the starch–chitosan blend films. Such phenomena could be due
to formation of intermolecular hydrogen bonding between
E (%) Thickness (mm) Moisture content (%) Tg (°C)
– 0.080 9.560 ± 0.456 68.08
13.537 ± 0.611 0.073 ± 0.004 20.916 ± 1.00 86.65
18.044 ± 0.624 0.157 ± 0.017 27.560 ± 0.4.26 77.24
14.022 ± 0.439 0.191 ± 0.024 28.868 ± 1.32 69.52
– 0.100 10.075 ± 0.25 72.15
– 0.030 18.978 ± 0.58 82.11
 H. Liu et al. / Journal of Food Engineering 116 (2013) 588–597 591

within the matrix causing micro-cracking on the surface when dry-

ing temperature is relatively high (65 ± 1 °C in the current study).
However, the antiplasticization effect seems only observed in the
2.5% glycerol, rather than the reported ‘threshold’ of glycerol,
No Glycerol
12% in starch only system. such finding could be resulted from
2.5% Glycerol
the addition of chitosan which strongly interact with glycerol and
Intensity (a.u.)

starch. Such interaction affected the threshold of glycerol in the

5% Glycerol
starch–chitosan polymeric network. The fact that no antiplasticiza-
10% Glycerol tion (observed as visible cracking in SEM image) occurred in 5% (w/
w) and 10% (w/w) glycerol-containing samples might suggest that
the hydrogen bonding between starch and chitosan suppressed the
Plain Starch antiplasticization behavior in wider glycerol range as suggested in
the case of starch–glycerol blends. Above the possible threshold
1% Chitosan glycerol concentration of 2.5% (w/w), the formation of smooth
and homogenous surface in the film suggests to the formation of
5 10 15 20
o
25 30 35
plasticized and rubbery starch–chitosan–glycerol films (Fig. 2).
2 Theta ( C)
Admittedly, only one sample (2.5% glycerol) is not sufficient to de-
Fig. 1. The X-ray diffraction patterns for the plain starch, pure chitosan and starch– fine the threshold for this effect, more experiments will be further
chitosan blend films at different glycerol concentrations. carry out to investigate this effect.

Table 2
The position of main X-ray diffraction peaks of different film samples.
3.5. Starch/Chitosan–glycerol interactions

Sample ID Peak position (°)
No glycerol 5.25 14.9 16.75 19.2 21.35
2.5% Glycerol 5.05 16.6 19.2 21.35
5% Glycerol 5 16.6 19.4
10% Glycerol 19.7
Plain starch 5.9 14.95 17.6 19.65 22.21
1% Chitosan 9.4 18.55
chitosan and starch which did not favor the crystallization of
starch and altered the chitosan structure (disappearance of the dif-
fraction peak at 9.4°). Similar observations were made by Xu et al.
who suggested that increasing the amount of starch in chitosan
film suppressed the crystalline peak of chitosan due to the change
in structure resulting from the interaction between chitosan and
starch (Xu et al., 2005). Such results had also been confirmed to ex-
ist in other polysaccharide systems such as cellulose (Hasegawa
et al., 1992; Jia-hui et al., 1999; Zhai et al., 2004). We also observed
that the crystalline peaks of plain starch shifted slightly to lower
degrees when chitosan was added (Table 2). This could be consid-
ered as further evidence of the formation of hydrogen bond be-
tween starch and chitosan.
The role of glycerol on the rubbery-crystalline characteristics of
starch–chitosan film was investigated by comparing the diffraction
patterns of films containing different concentration of glycerol (no
glycerol, 2.5%, 5% and 10% (w/w) glycerol). The extent of crystallin-
ity of the starch–chitosan films decreased when the concentration
of the glycerol increased (Table 2). The decrease in the degree of
crystallinity can be deduced or evidenced from the disappearance
of several peaks in the films containing high glycerol (10%, w/w)
concentration. This film has only one diffuse peak located at
19.7° (Fig. 1).
3.4. The surface morphology of the films
It is interesting to note that the SEM image of the starch–chito-
san film containing 2.5% (w/w) glycerol is different from the
images of other films (Fig. 2). This film exhibited cracking on the
surface. Considering the fact that the drying conditions for all these
films were identical (65 ± 1 °C and mild air flow), it can be con-
cluded that the relatively low concentration of glycerol (2.5%)
might have led to the anti-plasticization effect of glycerol. When
the glycerol concentration is low, the typical anti-plasticization ef-
fect of glycerol occurs and results in tighter polymeric network
Crystallinity (%) The FTIR spectra of the starch–chitosan blend films are shown
13.65 ± 0.03 in Fig. 3. The FTIR spectra of starch-only and 1% (w/w) chitosan
14.81 ± 0.11 films are also included for comparison purpose.
11.91 ± 0.02
The typical region of saccharide bands covers 1180–953 cm1
9.53 ± 0.03
15.83 ± 0.09 which is often considered to comprise vibration modes of C–C
9.75 ± 0.07 and C–O stretching and the bending mode of C–H bonds (Fig. 3).
These bands turned out to be the most intensive absorbance in
the IR-spectra (Fig. 3). As shown in Fig. 3, additional bands at
3300 cm1, 1630 cm1 and broader peaks centered at 2200 cm1
were also observed, which were typical bands associated with indi-
vidual biopolymer components in addition to the contribution of
the water absorption (Souza and Andrade, 2002).
For pure chitosan film there are four main characteristic absorp-
tion bands. The first one is a broad band ranging from around
3600–3100 cm1 which is attributed to N–H and OH–O stretching
vibration. This band is also, to some extent, contributed by the
intermolecular hydrogen bonding of chitosan molecules (De
Vasconcelos et al., 2006; Jiang et al., 1997). Second one, the two
weak bands located at 2884 cm1 and 2872 cm1 are from CH
stretching (Zivanovic et al., 2007). Third one is the amide-I band lo-
cated around 1640 cm1 (Wan et al., 2006). The last one, the bond
around 1550 cm1 is the amide-NH2 absorption band (Duan et al.,
2004). In the case of starch-only film, the typical absorption band
at 3300 cm1 is attributed to the hydrogen-bonded hydroxyl
groups associated with free inter and intramolecular bonded hy-
droxyl group. The third band at 1642 cm1 is assigned to the
d(O–H) bending of water (Mano et al., 2003). Finally the bands at
1150, 1077, 1009, and 931 cm1 are resulted from the C–O in C–
O–H and C–O in C–O–C chemical bonds, respectively (Jiugao
et al., 2005).
The interaction occurring in a specific system can be uniquely
reflected in the changes of the spectral peaks wavenumbers (Yin
et al., 1999). As can be seen from Fig. 3, compared to the IR spectra
of reference samples (pure chitosan film and starch-only film), the
amide-I and NH2 characteristic peak of chitosan in the IR spectra of
starch–chitosan blend film shifted from 1553.77 cm1 to
1556.47 cm1 and 1638.68 cm1 to 1638.97 cm1, respectively.
These results amply suggested that interactions had taken place
between the hydroxyl groups of starch and the amino groups of
chitosan (Meenakshi et al., 2002; Xu et al., 2005). Furthermore,
the characteristic peak of inter and intra-molecular hydrogen
bonds in starch (3326.98 cm1) and chitosan (3267.82 cm1)
shifted to a higher wavenumber at 3328.36 cm1. This shift
592 H. Liu et al. / Journal of Food Engineering 116 (2013) 588–597
Fig. 2. SEM images for the plain starch, pure chitosan and starch–chitosan blend films at different glycerol concentrations. All bars: 10 lm.
indicated the formation of the inter and intra-molecular hydrogen
bonding between chitosan and starch.
The effect of glycerol can be analyzed by comparing the spectra
of glycerol-containing films to the spectra of starch–chitosan blend
films without glycerol. As shown in Fig. 3, the main peaks located
at 3328.36, 2927.63, 1638.97 cm1 of the glycerol free film have
shifted to higher wavenumbers after the glycerol was added. This
indicated that the addition of glycerol promoted the hydrogen
bonding interactions among chitosan, starch and glycerol.
In the polysaccharides and polyol system, hydrogen bonding
changes are of great importance. Furthermore, in the FTIR spectra,
the most direct method to distinguish the molecular interaction is
to monitor the band shifts of certain function groups. In the current
study, both the chitosan and starch are gigantic molecular with
complex structure and stereo configuration. Therefore, in the glyc-
erol involved interaction, the hydrogen associate functional group
such as (–CH, –OH) is believed to be a more reliable indicator to
clarify its relevant interaction of glycerol with other component.
When the IR spectra of films containing different percentage of
glycerol (2.5%, 5.0% and 10%, w/w) are compared, it can be seen that
the peaks at 3345.28 cm1 and 2887.54 cm1 shifted slightly to-
wards the lower wavenumber (Fig. 3b). This indicated that slightly
weakening of the glycerol associated hydrogen bonding had taken
place (lower wave number). Such result is expected considering
the hydrophilic nature of all the materials (glycerol, starch and
chitosan) involved. When the glycerol concentration in the matrix
is increased, more –OH groups are available for starch–glycerol
and/or chitosan–glycerol interactions, which results in slight weak-
ening of glycerol–glycerol interactions (reflected in the shifting of
2887.54 cm1 band to a lower wave number of 2885.64 cm1 (5%
(w/w) glycerol) and 2884.24 cm1 (10% (w/w) glycerol)). Such
hypothesis is well agreed with the concept of anti-plasticization ef-
fect caused by low-amount of plasticizer where the addition of
plasticizers weakened strength of macromolecular interactions
(Talja et al., 2007). In other words, the shift (to a lower wavenum-
ber, means interaction weakened) of the –OH associate band at
3345.28 cm1 when increasing the glycerol amount from 2.5% to
10%, was well comply with the finding that 2.5% sample showed
anti-plasticization behavior in surface morphology and mechanical
section. Furthermore, the disappearance of the amide-NH2 absorp-
tion band in all the films containing glycerol suggested that strong
interactions occur between glycerol and chitosan even at a low
plasticizer concentration (2.5%, w/w). Furthermore, the existence
of the amide-NH2 absorption band in the no-glycerol sample fur-
ther supported the above argument (Fig. 3).
3.6. The glass–rubber transition of the films
As shown in Table 1, the Tg values of two reference films
(starch-only and pure chitosan films) are 72.2 and 82.1 °C, respec-
tively at corresponding moisture content values of 10.08% and
18.18% respectively. The Tg value for the starch–chitosan blend
(no glycerol) film was lower compared to both the reference films.
In other words, less energy was required for the blend film to drive
the polymer chain to be plasticized. These results supported the
SEM observation that the chitosan microdomains were homoge-
neously dispersed within the starch matrix when these two com-
ponents were blended.
Comparing the results across the blend films, increasing the
glycerol concentration decreased the Tg of the films from
86.65 °C (2.5% (w/w) glycerol) to 77.24 (5% (w/w) glycerol) and
69.52 °C (10% (w/w) glycerol), respectively (Table 1). Such results
are consistent with the commonly known plasticization effect of
glycerol on the Tg of the polysaccharide systems (Lourdin et al.,
1997a,b; Pouplin et al., 1999). However, the Tg values of 2.5% glyc-
erol sample was found to be higher than those of starch-only and
pure chitosan films. The antiplasticized state of the films can also
be corroborated from SEM observations that micro cracking were
observed in the surface of the 2.5% (w/w) glycerol film which re-
flected the brittle/rigid property of thie film. A possible reason
for this ‘antiplasticization’ behavior could be the strong interaction
between the -NH3 group of chitosan and the hydroxyl groups of
glycerol. The presence of such interaction was evidenced by the
disappearance of the chitosan amide-NH2 adsorption band
(1553.77 cm1) associated band in the starch–chitosan–glycerol
films. Strong hydrogen bonding formed between chitosan and
glycerol strengthened the polymeric network thereby increasing
the Tg values in the corresponding samples. Also, considering the
low glycerol content (2.5% and 5%, w/w), both of these concentra-
tions are lower than the reported antiplasticization threshold for
glycerol(12%) (Lourdin et al., 1997a,b). The fast drying process
(65 °C) could partly freeze the polymeric network without giving
chance for water molecules within the film matrix to equilibrate
during the film formation process. Moreover, the films were stored
at a low aw of 0.113 which is expected to increase the moisture loss
to rather than moisture gain from the storage headspace. Thus, it is
 H. Liu et al. / Journal of Food Engineering 116 (2013) 588–597 593

(a)
3326.98

3326.98 2927.55
2927.55

1642.70
1642.70

3267.82
3267.28
Plain Starch
2872.84
2827.84 1553.77
1553.77
Absorbance (%)

1638.68
1638.68

1% Pure Chitosan
3298.97
3298.97

2932.23 Typical saccharide bands region

2932.23
2880.33
2880.33 (1180cm-1-953-1)

Pure Glycerol 1416.98

4000.2 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600
Wavenumber (cm-1)

(b) Typical saccharide bands region

(1180cm-1-953-1)
3336.82 2884.24
2884.24
2935.64
2935.64
3336.82
1650.63
1650.63
3339.57
3339.57
10% Glycerol

2885.64
2885.64
2932.56
2932.56

1650.27
1650.27
Absorbance (%)

3345.28
3345.28
5% Glycerol

2887.54
2887.54
2930.97
2930.97
1649.43
1649.43
3328.36
3328.36 2.5% Glycerol

2927.63
2927.63
1556.47
1556.47
1638.97
1638.97

No Glycerol

4000.2 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600
Wavenumber (cm-1)

Fig. 3. The FTIR spectra for the prepared samples; (a) plain starch, 1% pure chitosan film and pure glycerol and (b) starch–chitosan blend films at different glycerol
concentration (No Glycerol, 2.5%, 5% and 10% (w/w) glycerol.).

expected that the moisture migration into the film is prevented
which greatly reduces the probability of lubricating of the poly-
meric network by the absorbed moisture. In polyol plasticized
polysaccharide polymeric systems, similar finding was observed.
Chaudhary et al. (2011) reported high Tg values at low plasticizer
content in extruded starch–glycerol composite material when
equilibrated at low moisture contents. Hence, the addition of glyc-
erol at low concentration could lead to a more strengthening rather
than softening of polymeric network.
3.7. NMR analysis
3.7.1. 1H NMR analysis
Hydrogen bond is the most important bond in polysaccharides
and the interactions associated to it are complex and hardly well
documented due to the intricate nature of such molecules. The
pure chitosan film did not dissolved well in the DMSO-d6, thus
the spectra of pure chitosan film is not presented in this study.
All the peaks are calibrated with the peak position of DMSO-d6 at
2.54 ppm (Liu et al., 2011a,b). The 1H NMR spectra of the reference
samples (starch-only and pure glycerol) are presented in Fig. 4a.
As shown in Fig. 4a, the main characteristic peaks for gelati-
nized starch-only are partially assigned (inset of Fig. 4a). The peak
positions were slightly higher than the reported values in Liu
et al.’s publication on extruded gelatinized starch samples (Liu
et al., 2011a,b). The interactions could be studied by comparing
the shift in peaks and intensity of the reference components
(starch and glycerol) with those of blend film from the NMR spec-
tra under the identical same condition. When the chemical envi-
ronment of hydrogen is changed, the electron distribution in the
594 H. Liu et al. / Journal of Food Engineering 116 (2013) 588–597

water with its mobility in pure glycerol. For example, the peak position
(a)
shifted to a higher frequency from 4.48 ppm to 4.523 ppm,
4.528 ppm and 4.541 ppm for 2.5%, 5% and 10% (w/w) glycerol
samples, respectively. These observations suggested that the
mobility of the glycerol molecules slightly decreased with the in-
n crease in the glycerol concentration due to stronger glycerol–
Assign. Shift (ppm)
A 5.548
starch/chitosan interactions which restricted the glycerol mobility
B 5.445 compared to its mobility in the glycerol sample. When a peak is
C 5.148 shifted to a higher frequency, more energy is necessary to excite
D 4.627
the proton from one spin state to another compared to those pro-
PARTIALLY ASSIGNED. tons at low frequency. In other words, the shift to a higher fre-
AB C D DMSO-d6 quency for a peak indicates that the electron density increases
resulting from a more chemically restricted environment (more
firmly bonded) surrounding the atoms. These observations were
corroborated by the FTIR spectra (Section 3.5) which showed that
the presence of glycerol promoted the formation of hydrogen
(b) 10% Glycerol bonds among glycerol, starch and chitosan. The increased interac-
tions among these three components brought about by hydrogen
bonds were reflected in shifting of glycerol’s characteristic peak
(2887 cm1) to a lower wave number (2885 cm1 and
2884 cm1 for 5% and 10% (w/w) glycerol, respectively). More-
over, it can be seen that the intensity of these peaks (5.5 ppm,
etc.) was found to gradually decrease when the concentration of
5% Glycerol the glycerol increased. This observation also indicated that the
mobility of the proton in starch chain was progressively re-
stricted. This phenomenon can be explained on the basis of
hydrogen bonding interactions occurring between starch–chito-
san–glycerol films.
2.5% Glycerol Similar observations were made regarding the shift in –OH
group’s characteristic peak positions of starch chain. Three main
peaks associated with –OH group occurring at 5.548, 5.449,
4.627 ppm shifted slightly to higher frequencies (Table 3) due to
the hydrogen bonding interactions. However, it should be noted
that, the magnitude of this shift from glycerol was larger than that
from the starch peaks. This phenomenon can possibly be attributed
Fig. 4. (a) 1H spectra for plain starch and the assignment of main peaks and (b) 1H to the large molecular size and complicated stereo-configuration of
spectra for 2.5%, 5.0% and 10% (w/w) glycerol films and the assigned main peak starch when compared to the highly mobile glycerol molecules.
positions.

molecules will be altered and such changes directly result in shift
of the peak position obtained in a proton NMR measurement
(Becker, 2000). The main characteristic peaks for reference films
and the starch–chitosan blend film are summarized in Table 3.
The 1H NMR spectra for starch–chitosan–glycerol blend films
are shown in Fig. 4b. In the solution state NMR study, the intensity
and the position of the peaks reflect the molecular mobility of the
component or whether or not the interaction had occurred (Liu
et al., 2011a,b).
As shown in Fig. 4b, the high intensity of 10% (w/w) glycerol
sample was mainly due to the presence of larger amount of the
high mobility component (glycerol), when compared with the rest
spectra. It can be seen from Fig. 4b that the broadening of the
glycerol characteristic peak, assigned at 4.48, 3.448 and
3.382 ppm has occurred which indicated that the mobility of
glycerol molecules in the blend film was restricted compared
3.7.2. 13C NMR analysis
The hydrogen bonding interactions in the starch–glycerol films
are also reflected in the 13C NMR spectra. As mentioned in the pro-
ton NMR section, the pure chitosan film did not dissolve well in the
DMSO-d6 solvent, thus no high resolution 13C NMR could be ob-
tained in the case of pure chitosan film. The 13C NMR spectra of
all the films (except pure chitosan film) are presented in Fig. 5a–c.
The characteristic peaks for gelatinized plain starch are as-
signed in Fig. 5a. It can be seen from this figure that the charac-
teristic peak values were slightly lower than the reported values
for extruded high amylose starch (Liu et al., 2010; Liu et al.,
2011a,b). The two sharp peaks with high intensity in the spec-
trum of 2.5% (w/w) glycerol sample was assigned to be the char-
acteristic peaks from glycerol component due to its small
molecular size (high mobility) when compared to the starch or
chitosan chain. Furthermore, it was found that the characteristic

Table 3
The main characteristic peaks for plain starch, pure glycerol, and starch–chitosan blend films at different glycerol concentration (0%, 2.5%, 5% and 10% (w/w) glycerol).

Calibrated at DMSO-d6 at 2.549 ppm

Sample ID Main characteristic peak positions (ppm) from high frequency to low frequency
No glycerol 5.549 5.459 5.150 4.631 3.701 3.625
2.5% glycerol 5.553 5.450 5.150 4.628 4.523 4.512 4.444 3.70 3.63
5% glycerol 5.556 5.451 5.148 4.631 4.528 4.518 4.451 3.70 3.624
10% glycerol 5.558 5.454 5.148 4.640 4.541 4.529 4.464
Plain starch 5.548 5.449 5.148 4.627 3.697 3.624
Pure glycerol 4.48 3.448 3.382
 H. Liu et al. / Journal of Food Engineering 116 (2013) 588–597 595

(a) (b) (c)

63.67ppm
63.05ppm

1 100.66ppm
2 79.29 ppm High intensity peaks from
3 73.79 ppm high mobility glycerol.
4 72.52 ppm
5 72.18 ppm
6 61.05 ppm

72.37ppm
73.09ppm

Low intensity peaks from

low mobility
polysaccharides which
decreased upon increasing
glycerol concentrations.

Plain Starch Pure Glycerol Starch-Chitosan (2.5%Glycerol)

(d)

Fig. 5. 13C NMR for (a) plain starch, (b) pure glycerol, (c) 2.5% (w/w) glycerol film and the assigning of characteristic peak positions, and (d) Comparison 13C NMR for 2.5%, 5.0%
and 10% glycerol films and assigning of the characteristic peak positions.

peaks of glycerol shifted to a higher frequency (Fig. 5b). These
values shifted from 72.37 ppm and 63.05 ppm (pure glycerol) to
73.09 ppm and 63.67 ppm, respectively. Such observations mutu-
ally supported the findings obtained from the proton NMR analy-
sis that the addition of glycerol promoted the hydrogen bonding
interactions in starch–chitosan polysaccharide system by travel-
ling among starch–chitosan polymeric network and forming
hydrogen bonds.
The comparison 13C NMR spectra of films containing different
amount of glycerol are presented in Fig. 5d. The intensity of the
starch characteristic peaks decreased gradually when the concen-
tration of glycerol increased. This observation also indicated that
the starch and chitosan chains were more firmly bounded by the
hydrogen bonding interactions occurring at a high-glycerol con-
centration. Therefore, the formation of better ordered and stronger
polymeric network was expected in the films containing higher
concentration of glycerol (within the concentration tested). Thus
the increase in free volume in the films containing higher concen-
tration of glycerol (10%, w/w) was reflected in the observed low Tg
value (Table 1).
596 H. Liu et al. / Journal of Food Engineering 116 (2013) 588–597
4. Conclusions
The effects of glycerol concentration (0%, 2.5%, 5% and 10%, w/
w) on the mechanical and thermal properties and molecular level
interactions in starch–chitosan films were comprehensively stud-
ied. It can be concluded that the antiplasticization effect was ob-
served in starch–chitosan–glycerol films when the glycerol
concentration was 2.5% (w/w). The antiplasticization was reflected
in visible surface cracking in the SEM image and the high Tg value
of 2.5% (w/w) glycerol film. At glycerol concentration of 5% (w/w)
and above the starch–chitosan–glycerol films were typically plasti-
cized by glycerol as evidenced from the significant (p < 0.05) de-
crease in both tensile strength and Tg.
The addition of glycerol promoted the interactions among
chitosan, starch and glycerol through hydrogen bonding. This
was reflected as the main peaks located at 3328, 2927,
1638 cm1 of the starch-only and pure chitosan films have shifted
to higher wavenumbers in the presence of glycerol. The stronger
glycerol–starch/chitosan interactions in samples containing higher
glycerol concentration were confirmed from the FTIR (characteris-
tic band shifted to a higher wavenumber) and NMR results (de-
crease of glycerol mobility in corresponding samples). The
intensity decrease in the glycerol related peaks in both 1H NMR
and 13C NMR indicated the strong interactions occurring among
starch, chitosan and glycerol.
Acknowledgments
The authors are grateful to Bruce Armstrong, Delina Muscat and
Amir Ghandi for their help in some of the experiments. Stafford
McKnight is gratefully acknowledged for helping with SEM and
XRD experiments. Prof. Shin-ichi Yusa is gratefully acknowledged
for his fruitful inputs on NMR experiments. This project was sup-
ported by University of Ballarat led Collaborative Research Net-
work (CRN).
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